Alcohol use disorder (AUD) is a major public health concern characterized by the loss of control over alcohol drinking and the emergence of negative emotionality during abstinence from alcohol. The transition to alcohol dependence is associated with the concomitant dysregulation of executive function by the medial prefrontal cortex (mPFC) and the recruitment of corticotropin releasing factor (CRF) and its CRF receptor 1 (CRF1) in the central nucleus of the amygdala (CeA), but much less is known about the role of CRF/CRF1 signaling in cortical areas. Critically, the development of new tools to access subpopulations of CRF1 neurons has facilitated our studies of function and connectivity of CRF/CRF1 systems. In the original grant, we use an innovative transgenic mouse models in which CRF1 expressing (CRF1+) neurons co-express green fluorescent protein (GFP) (CRF1:GFP mice) to understand the cell type-specific effects of acute and chronic alcohol in local and downstream CeA microcircuits. Preliminary data have revealed that the CRF1+ neurons are highly expressed in mPFC layer 2/3 and undergo specific neuroadaptations following chronic alcohol compared to unlabeled (CRF1-) neurons. Thus, in this renewal, we will continue to use CRF1:GFP and CRF1:Cre mice to characterize the electrophysiological, neurochemical and morphological properties of both CRF1+ and CRF1- neurons in the mPFC, and elucidate the role of mPFC CRF1+ circuitry in anxiety-like and drinking behaviors. Through a systems biology approach, we will delineate the CRF1-dependent effects of ethanol in mPFC, as well as amygdala-mPFC connectivity that may regulate ethanol dependence and withdrawal and contribute to associated anxiety-related behaviors. In addition, we will use fluorescence activated cell sorting (FACS) followed by whole transcriptome analysis to reveal molecular signatures and neuroadaptations of this specific CRF1 cortical population, which may identify new promising targets for treatment strategies for alcoholism.